Microtome sectionable gel support structure and methods

ABSTRACT

A sectionable tissue sample support structure including a gel compound formed into a self supporting geometric shape for retention and orientation of at least one tissue sample during a histopathology process including processing, embedding and microtome slicing of the tissue sample. A method of orienting, processing, embedding and microtome slicing a tissue sample using a gel compound preformed into a self supporting geometric shape. A combination including the sectionable tissue sample support structure and a package containing the sectionable tissue sample support structure.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of application Ser. No. 17/015,813filed Sep. 9, 2020 (pending) which is a divisional of application Ser.No. 14/011,809 filed Aug. 28, 2013 (now U.S. Pat. No. 10,794,804), whichclaims the priority of Application Ser. No. 61/700,062 filed Sep. 12,2012 (expired), the disclosures of which are hereby incorporated byreference herein.

TECHNICAL FIELD

The present invention relates generally to histopathology and, morespecifically, to devices and methods related to the processing,embedding and microtome sectioning of biological tissue samples forpurposes of scientific or medical examination.

BACKGROUND

Histopathology is the science of preparing tissue samples formicroscopic examination by a pathologist. The ultimate purpose is todiagnose the medical condition of a patient from whom the tissue samplewas taken. One or more tissue samples are treated in a processing stepto remove fluid from the tissue samples and replace the fluid with mediasuch as paraffin. The tissue samples are then embedded in media,typically paraffin, and formed into a block. The paraffin block togetherwith the embedded tissue sample(s) is sliced into very thin sectionswhich are then adhered to a glass microscope slide. The tissue sectionsare stained and prepared for microscopic examination by a pathologist.

One of the most crucial steps in the process is to properly orient thetissue samples in relation to the sectioning plane during the paraffinembedding process. Large sections of organs or tumors may or may nothave specific orientation requirements when embedded in the paraffinblock. However, most tissue samples have some requirement fororientation. Techniques or products have become available to help thehistotechnologist obtain the proper orientation of the tissue sample inan efficient manner. Gels are available to assist with orientation,however, these are typically two-part gels which need to be mixed orfixing gels which either need to be cooled or activated to then hold thetissue in the proper orientation. Therefore, these uses of gel requireadditional time and special techniques to obtain a desired tissueorientation. In addition, modern techniques such as those described inU.S. Pat. Nos. 7,156,814; 7,722,810; 7,776,274; and 8,034,292 and8,383,067, (the disclosures of which are hereby fully incorporated byreference herein), are examples of efforts to accommodate specific typesof tissue samples and their need for orientation. Some tissue types aremuch more difficult to embed properly than others. This is especiallytrue for small tissue fragments. Automation of the histopathologyprocess provides further challenges because manual embedding methods forsuch small tissue samples cannot keep pace with the throughput of thelaboratory.

Histopathology laboratories, like all businesses, are under pressure tobecome more cost effective while maintaining high quality and shorterturnaround times for pathology reports. Tissue sizes and shapes withcorrespondingly intricate and unique orientation requirements make itvery difficult to meet these demands. Even within advanced automatedprocesses some tissue types continue to need extra care and attentionfor proper embedding. Until automation of the paraffin embedding andmicrotome sectioning process these steps were largely carried out inmanual steps by trained histotechologists. While some examples of“embedding aids” are noted in the prior art, all fall short ofaddressing this need at least for a certain subset of tissue types. Forexample, there are very small tissue samples that are routinely examinedby histopathology process. These samples can be so small thatmanipulation with forceps is difficult and orientation is extremelydifficult. To compound this problem the samples are sometimes so smallthat special types of cassettes are needed to be certain that the tissuesamples do not escape from their tissue processing cassettes.

Small thread-like strands or indiscriminate chips or scrapings from abiopsy procedure are particularly difficult to maintain in properorientation during the embedding process. Therefore, in histopathologythere are tissue types and procedures which require special handling andembedding technique due to their size and orientation requirements.These procedures slow down the workflow, and are subject to a poorquality outcome. In addition to human pathology, the histopathology isused to diagnose disease in non-human species. For instance,pharmaceutical companies commonly use rodent models to detect druginteraction or side effects prior to any testing on humans. One rodentmodel commonly used is a mouse. Genetic engineering has allowedcompanies to design mice which are very sensitive to certain types ofdisease, therefore speeding up drug development. These companiesroutinely use the mouse model to observe drug effects, prior toproceeding to costly trials. Histopathology confirms the effects of thedrugs through examination of affected tissue types. Hundreds ofthousands of mice are studied every year. An example of one suchstructure is the central nervous system. Limbic, brain stem, spinal cordand optic nerve tissue are most indicative of abnormal growth orpathology for some drugs. As one can imagine, some nerve threads from amouse are exceedingly small. One structure in particular, the opticnerve, is ½ mm in diameter and less than two mm long. It is asignificant challenge to hold tiny tissue samples upright in a dropletof paraffin while the droplet cools and solidifies, or while a two-partgel solidifies around the optic nerve tissue sample. Often times, whentrying to release the tissue sample, the sample sticks to the forcepsinstead of the embedding media. This is a tedious process that requiresextreme dexterity, training and experience to obtain consistent, highquality results. This coupled with the need to work efficiently hascreated a need for a small tissue holder that provides significantadvantages to those in the field.

SUMMARY

In one embodiment, a sectionable tissue sample support structurecomprises a gel compound formed into a self supporting, threedimensional geometric shape for retention and orientation of at leastone tissue sample during a histopathology process which includesprocessing, embedding and microtome slicing of the tissue sample. Thetissue sample support structure may have one or more additional featuresas described herein, with examples summarized below.

The gel compound may be resilient such that after deformation from anoriginal shape, the gel compound reverts back to the original shape.This can assist with various uses, such as tissue sample retention. Thesectionable tissue sample support structure can further comprise atissue sample receiving space formed into the gel compound for retainingthe tissue sample during the histopathologic process. As examples, thereceiving space may comprise at least one of: a slit, a hole, a recess,or combinations thereof. The sectionable tissue sample support structurecan further comprise a tissue retaining structure configured to retainthe tissue sample in the tissue receiving space. For example, the tissueretaining structure can take the form of at least one deformable portionof the gel compound configured to apply a force to the tissue sample andthereby retain the tissue sample in a desired orientation. Variousstructures, such as jaws or flaps of the gel structure, may be used asthe retaining structure. The deformable portion can be a hinged jawelement configured to move between open and closed positions and apply aclamping force to the tissue sample in the closed position to therebymaintain the tissue sample in a desired orientation. The two portions ofthe gel compound that receive the tissue therebetween may be on the sameintegral piece of preformed gel or may be two separate pieces, forexample, that sandwich one or more tissue samples therebetween. Thereceiving space may be defined between flat or planar surfaces of thepreformed gel, or may include one or more three dimensional spaces, suchas grooves, slots or recesses in the preformed gel structure that holdthe tissue sample(s). The gel compound is permeable to fluids andreagents used in processing the tissue sample. This aspect of the gelcompound ensures that full cross-sectional preservation of the tissuesample is achieved during tissue processing with conventional fluids andreagents. A prepackaged sectionable tissue sample support structurecomprises a gel compound as set forth in any of the description hereinand a package enclosing the gel compound. The packaging may provideclean or even sterile conditions for the gel compound, and also at leastassist with retaining moisture within the gel compound to maintainresiliency of the gel structure. The package may contain a suitablesolution for purposes of maintaining resiliency of the gel compound. Thegel compound and/or package may contain additives for preventing growthof mold, fungus or bacteria, for example. Alternatively, or in addition,the package may be subjected to various other types of non-contactsterilization, such as electron beam or gamma radiation.

The invention further provides methods for orienting, processing,embedding and microtome slicing a tissue sample using a gel compoundpreformed into a self supporting geometric shape. For example, onemethod comprises retaining the tissue sample in a desired orientationbetween first and second portions of the preformed, geometrically shapedgel compound. The tissue is processed while in the desired orientationby subjecting the tissue sample and the preformed, geometrically shapedgel compound to processing fluids and reagents. The preformed,geometrically shaped gel compound and the tissue sample are embedded inan embedding media while in the desired orientation to form a microtomesectionable block of the embedding media, the tissue sample and thepreformed, geometrically shaped gel compound. The microtome sectionableblock is then sectioned to obtain thin sections of the tissue sample fordiagnosis.

Another method comprises adhesively retaining the tissue sample in adesired orientation on the preformed, geometrically shaped gel compound.The tissue is processed while in the desired orientation by subjectingthe tissue sample and the preformed, geometrically shaped gel compoundto processing fluids and reagents. The preformed, geometrically shapedgel compound and the tissue sample are embedded in an embedding mediawhile in the desired orientation to form a microtome sectionable blockof the embedding media, the tissue sample and the preformed,geometrically shaped gel compound. The microtome sectionable block isthen sectioned to obtain thin sections of the tissue sample fordiagnosis.

The methods of the invention may have various other aspects or steps.For example, the method can further comprise removing the preformed,geometrically shaped gel compound from a package prior to suitablysecuring the tissue sample to the preformed, geometrically shaped gelcompound. Retaining the tissue sample between the first and secondportions can further comprise retaining the tissue sample betweenresiliently deformable portions of the preformed, geometrically shapedgel compound. The first and second portions may be on the same threedimensional gel structure, such as the portions on either side of atissue receiving slit for example, or each may comprise separate gelstructures, such as two gel sheets between which the tissue sample isplaced. Retaining the tissue sample between the first and secondportions can further comprise retaining the tissue sample in a hole orrecess formed in the preformed, geometrically shaped gel compound. Therecess may, for example, be an elongate recess formed lengthwise alongan outer surface of the preformed, geometrically shaped gel compound.The method may further include securing the preformed, geometricallyshaped gel compound and the retained tissue sample on a tissue supportstructure at least prior to the embedding and microtome sectioningsteps. The tissue support structure can further comprise a microtomesectionable support structure, such as a cassette.

The invention provides various advantages and features that address thecomplications or challenges associated with current tissue sampleprocessing and embedding techniques. For example, the inventive gelcompound, which is formed into a self-supporting geometric shape, can bepreformed into any shape or configuration to facilitate specificembedding tissues or challenges. The compound can allow tissue samplesto be held fast in a desired orientation and to hold the tissue sampleor samples throughout tissue processing, embedding and sectioningprocedures. The tissue sample is not lost during the processingtechniques and is held securely and oriented precisely for subsequentembedding and sectioning without further manual or automated handling ofthe tissue sample itself. The tissue may be held in the desiredorientation during positioning by the user and this includesdisengagement of forceps from the tissue sample while the tissue sampleis engaged with the gel compound. The tissue sample may be quickly andeasily engaged and retained by the preformed gel compound with standardtools or implements and requires no mixing, drying, cooling or otheractivation of the compound during the orientation procedure.

As mentioned above, the gel compound allows processing of the tissuesample as the tissue sample is held and retained in the desiredorientation. Thus, the compound is porous to fluids and reagents used toprocess the tissue sample. The gel compound does not interfere with thediagnostic process and, for example, if the gel compound absorbs orotherwise takes up the stain used on the microscope slide sections, thegel compound is distinguishable from the surrounding tissue sample.Preferably, the gel compound does not absorb the stain and, therefore,the gel compound is nondistracting to the user during the diagnosticprocess associated with the microscope slide holding one of the ribbonsections formed with the microtome. The three dimensional, preformed gelcompound, does not interfere with microtome sectioning of very thinsections of the embedding material, the gel compound, and the tissuesample. Therefore, high quality, very thin ribbons may be sectioned andplaced on glass microscope slides for diagnostic purposes. The threedimensional, preformed gel compound can serve to encapsulate and traptissue during tissue processing with chemical fluids and reagents, aswell as during embedding, sectioning and microscope slide preparation.As a result, tissue is not lost during these procedures. In addition,the invention results in little or no artifact being introduced into thetissue sample(s), which could interfere with proper microscope slidepreparation and diagnoses of the tissue sample(s). Finally, thesectionable tissue sample support structures, including the preformedgel compounds, may be used in conjunction with automated sectionablecassettes to speed the overall histologic procedure.

The gel compound may be manufactured into any number of physicalconfigurations based, for example, upon the needs of the pathologist orscientist, or based on the needs of the specific pathologic/scientificprocedure being performed. The gel compound may be supplied in bulk formfor the histotechnologist to make the desired tissue sample supportstructures. For example, the gel compound of this invention may beeither extruded or cast into a sheet form, and then cut intoappropriately sized sections for the intended application. For example,the preformed, three dimensional gel compound may be used in the form ofsmall blocks of any desired shape, or small sheets. In the case of sheetforms, the tissue sample(s) may be sandwiched between two of the sheets,or between one of the sheets and another element, such as another typeof sectionable support. Various features may be incorporated into thepreformed, three dimensional gel compound to assist with its use duringthe different procedures involved in histopathology. As another example,physical features such as recesses or grooves may be formed in one ormore surfaces of the three dimensional gel compound for express of fluidor fluid run-off as the tissue sample is placed on or secured to the gelcompound.

Various additional features and advantages will become readily apparentto those of ordinary skill in the art upon review of the followingdetailed description of the illustrative embodiments, taken inconjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective view of several preformed, sectionable tissuesample support structures constructed in accordance with a firstillustrative embodiment of the invention.

FIG. 2 is a perspective view of a single sectionable tissue samplesupport structure taken from FIG. 1 .

FIG. 3 is a perspective view illustrating the sectionable tissue samplesupport structure deformed or opened to receive tissue samples in areceiving space.

FIG. 4 is a perspective view illustrating the gel support structure ofFIG. 3 in a closed condition for securing the tissue samples in thereceiving space or slit of the gel support structure.

FIG. 5 is a perspective view illustrating the support structure of FIG.4 placed in a sectionable cassette, with the lid of the cassette in anopen condition.

FIG. 6 is a cross sectional view illustrating the cassette of FIG. 5 ina closed condition and embedded in a block of paraffin, as well ascoupled to a frame in preparation for a microtome sectioning operation.

FIG. 7 is a perspective view illustrating an illustrative embodiment ofa packaging system for the sectionable tissue sample support structureof FIG. 2 .

FIG. 8 is a cross sectional view of the packaged tissue sample supportstructures shown in FIG. 7 .

FIG. 9A is a perspective view of three sectionable tissue sample supportstructures formed in accordance with another illustrative embodiment,and contained in a device for allowing tissue sample support recesses orslots to be opened and closed.

FIG. 9B is a perspective view similar to FIG. 9A, but illustrating thedevice being used to open the recesses or slots for receipt of tissuesamples therein.

FIG. 10 is a perspective view illustrating the sectionable tissue samplesupport structures of FIGS. 9A and 9B receiving an adhesive containmentspray to further retain, seal and/or contain the tissue samples therein.

FIG. 11 is a cross sectional view taken through the sectionable tissuesample support structure of FIG. 10 to illustrate the adhesivecontainment layer used to seal and contain the tissue sample in place.

FIG. 12 is a cross sectional view similar to FIG. 6 , but illustratinguse of the sectionable tissue sample support structure of FIG. 11 .

FIGS. 13-16 are additional illustrative embodiments of tissue samplesupport structures constructed in accordance with further embodiments.

FIG. 17A is another illustrative embodiment of a sectionable tissuesample support structure having a receiving space in the form of a holecommunicating with respective slits.

FIG. 17B is a perspective view of the support structure of FIG. 17A, butwith a tissue sample contained in the hole.

FIGS. 18-20 are respective illustrative embodiments of additionalsectionable tissue sample support structures capable of being cut intomultiple pieces.

FIG. 21A is another illustrative sectionable tissue sample supportstructure receiving an elongate tissue sample.

FIG. 21B is a perspective view illustrating the sectionable tissuesample support structure of FIG. 21A holding respective tissue samplesand being sprayed with an adhesive containment spray for holding thetissue samples to the support structure.

FIG. 21C is a cross sectional view similar to FIGS. 6 and 9 , butillustrating use of the tissue sample support structure of FIG. 21B.

FIG. 22 is a perspective view of another sectionable tissue samplesupport structure constructed in accordance with an embodiment forholding various types of tissue samples.

FIG. 23 is a perspective view of another sectionable tissue samplesupport structure constructed in accordance with an embodiment forholding various types of tissue samples.

FIG. 24 is a perspective view of another sectionable tissue samplesupport structure constructed in accordance with an embodiment forholding various types of tissue samples.

FIG. 25 is a cross sectional view similar to FIG. 6 , but illustratinguse of the sectionable tissue sample support structure of FIG. 23 orFIG. 24 .

DETAILED DESCRIPTION

FIGS. 1-6 illustrate one of many possible forms for a sectionable tissuesample support structure constructed in accordance with the inventiveconcepts, and including a gel compound formed into a self-supportinggeometric shape. Throughout this specification, the term “block” may beused to describe various types of tissue sample support structuresconstructed with a gel compound and preformed into a geometric shape,but this term is not meant to be limited to any particular threedimensional geometric shape. Instead, the gel compound blocks may haveany shape, including the square or rectangular shapes shown, or blocksof any other curved, spherical, oblong or other shapes.

As used herein, a “gel compound” is defined as a dilute cross-linkedsystem, which exhibits no flow when in the steady-state, and includeshydrogels, organogels, and/or aerogels. The gel compounds are mostlyfluid, yet they behave like solids due to a three-dimensionalcross-linked network within the fluid. It is the crosslinkages withininternal components that give a gel compound its three-dimensionalstructure. In this way gel compounds are a dispersion of molecules of afluid within a solid in which the solid is the continuous phase and thefluid is the dispersed phase.

FIG. 1 illustrates a sheet 10 of extruded or cast gel compound formed,for example, to be 1.5 mm thick, 4 mm wide by 5 mm long. Individualsectionable tissue sample support structures or blocks 12 are formed andeach may have a dimension of 1.5 mm thick, 4 mm long, and 3 mm wide.These dimensions are merely illustrative and may be changed inaccordance with the needs of the user. The blocks 12 may be initiallyretained on a release paper or plastic tray 14, which may be part of apackage to be described below, and, as shown in FIG. 3 , thin,thread-like tissue samples 16 are retained in a tissue receiving spaceshown here as a slit 18 between two portions 20, 22 of the block 12. Oneportion 20 of the block 12 may be folded back or opened as shown in FIG.3 , and then closed as shown in FIG. 4 to secure the tissue sample 16 inthe desired orientation. The gel compound is resilient or elastic innature such that the opened portion 20 of the block 12 shown in FIG. 3resiliently closes against the opposite portion 22 to the retain thetissue sample(s) 16 between the two portions 20, 22. Alternatively, ifthe gel block 12 does not have sufficient elasticity or resilience, anadhesive compound or other means may be used to retain the two portions20, 22 in the closed position as shown in FIG. 4 . It will beappreciated that the tissue samples 16 are retained within the tissuereceiving space (i.e., a slit 18 in this embodiment), and the ends 16 aof the tissue samples 16 are flush against the other surface 12 a of theblock 12. In this manner, the ends 16 a of the tissue samples 16 will bepositioned and oriented correctly for embedding and microtome sectioningprocedures, as described below. In this regard, the outer surface 12 aadjacent to which the tissue sample is exposed will face the sectioningplane such that as sections are taken with a microtome blade, thesections will include thin cross sections at, and then inward of, thetissue sample ends 16 a.

One manner of processing, embedding and microtome sectioning of thetissue sample will be understood from a review of FIGS. 5 and 6 . Inthese figures, a cassette 30 is used as described, for example, in U.S.Pat. No. 5,817,032 (the '032 patent), or the other patents and publishedapplication incorporated by reference above. As the procedure will befully understood by reference to the '032 patent, as well as the otherpatents and published application incorporated by reference herein,additional disclosure is not necessary except to the extent appropriatefor an understanding of the present invention. As shown in FIG. 5 , thegel block 12 with the tissue samples 16 retained therein is placed intothe sectionable cassette 30. The lid 32 of the cassette 30 is thenclosed against one surface 12 b of the gel block 12 while the bottom 34of the cassette 30 engages the opposite surface 12 a of the gel block12, as best illustrated in FIG. 6 . Both the gel block 12 and the tissuesamples 16 are retained in the cassette 30 during processing of thetissue samples 16 which involves submerging of the sectionable cassette30, gel block 12, and the retained tissue samples 16 in various fluidsand reagents designed to extract the bodily fluids from the tissuesamples 16 and replace those fluids with, for example, paraffin. Afterprocessing, the sectionable cassette 30, and the gel block 12 with theretained tissue samples 16 secured within the cassette 30 or otherwisesecured to a suitable support, is secured within a frame 36 in theposition shown in FIG. 6 , and the frame/cassette assembly is placedinto a mold (not shown). Embedded material, such as paraffin, is thendirected into the mold through the frame 36 and the perforate cassette30 such that the paraffin takes the form of mold and solidifies into aparaffin block 38 as shown in FIG. 6 . Microtome sections are then takenby a microtome blade operated by a histotechnologist, as generallydescribed in the '032 patent, by facing off the bottom 34 of thecassette 30 until the tissue sample 16 is reached. At this point, verythin ribbon-like sections will be taken of the paraffin block 38, thegel block 12 and the tissue samples 16. Those ribbons (not shown) arethen placed on glass microscope slides (not shown) for examination anddiagnostic purposes.

FIGS. 7 and 8 illustrate one method and form of packaging the gel blocks12 shown in FIGS. 1-4 . In this example, the gel blocks 12 are placedonto a suitable support, such as a plastic tray 40, or wax coated paperor cardboard, and placed into a package which may take the form of a bag42 having a suitable fastening means such as a slide connection 44 forsealing the bag but allowing resealing as well. The bag 42 may be sealedin a clean or even sterilized condition and preferably holds the gelblocks under moisture proof or at least moisture resistant conditionsuntil use. Before sealing the bag 42, moisture may be added in anysuitable manner for purposes of maintaining resilience of the gel blocks12 during transport and storage. Additives may also include anti-fungal,anti-mold and/or anti-bacterial compounds. The user can remove the gelblocks 12 by sliding the paper or cardboard tray 40 out from the bag 42and peeling the gel blocks 12 off of the paper or cardboard 40. The gelblocks 12 have a tackiness that is inherent and allows them to beadhered to the paper or cardboard 40 but easily peeled off for use.

FIGS. 9A and 9B illustrate a device 50 that may or may not be part of apackaging structure for the gel blocks 12′. The device 50 has sidewalls52, 54 which removably retain the gel blocks 12′ therebetween. Thesidewalls 52, 54 may be compressed or squeezed together as schematicallyillustrated in FIGS. 9A and 9B. This opens respective slits 56 in eachgel block 12′ such that small pieces of tissue, such as threadliketissue samples 16 may be easily inserted in a vertical manner withineach open slit 56. Thus, the tissue samples 16 will be retained withinthe tissue receiving spaces or slits 56 of the gel blocks 12′substantially as shown and described with regard to FIGS. 3-6 . Once thetissue samples 16 are placed and retained into the slits 56, the gelblocks 12′ may be removed from the device 50 and used in accordance withthe above description, for example, regarding FIGS. 5 and 6 .

Alternatively, the gel blocks 12′ may be removed from the device and thesurface 12 a′ of the gel block 12′ that contains the slits 56 may besprayed with an adhesive or other coating material for purposes offurther retaining the tissue samples 16 within the gel block 12′. Asshown in FIGS. 10 and 11 , this sprayed coating of material 58 may beused to create an adhesive or coating layer 60 retaining the tissuesample 16 within the gel block 12′. This sprayed material may, forexample, take the form of a spray form of the gel itself or any suitableadhesive such a polyvinyl acetate (PVA), ethylvinyl acetate (EVA) orcyanoacrylate (CA) adhesives and should generally have the sameproperties as the gel material forming the block 12′ such as, forexample, porosity, stain resistance, etc., as discussed above. Asfurther shown in FIG. 12 , the gel block 12′ is placed into a cassette30 generally as described above in connection with FIG. 6 , such thatthe adhesive or other coating layer 60 faces the sectioning plane,defined by the bottom surface or wall 34 of the cassette and the tissuesample end 16 a also faces the sectioning plane. In this manner, afterthe bottom 34 of the cassette 30 is sectioned or faced off, themicrotome will begin sectioning the paraffin block 38 and the gel block12′, in addition to the tissue sample 16, as described above.

FIGS. 13-16 illustrate various other configurations of gel blocks,constructed with a compound having the formulations and properties asdescribed herein, but having tissue receiving spaces of various forms.FIG. 13 illustrates a gel block 70 with a tissue receiving space 72 inthe form of an oblong, elongate recess, while FIG. 14 illustrates a gelblock 80 with a pair of oblong holes or recesses 82 for receiving andretaining tissue samples 16. FIG. illustrates a gel block 90 with twoelongate square shaped recesses 92, while FIG. 16 illustrates a gelblock 100 with a single elongate recess 102, for example, holding a flatpiece of tissue 104 such as skin tissue oriented on edge relative to themicrotome sectioning plane.

FIGS. 17A and 17B illustrate another gel block 110 with an alternativetissue receiving space in the form of a central hole 112 and slits 114,116 extending from opposite sides of the hole 112. This hole 112 isconfigured to hold a cylindrical piece of tissue, such as a tubularpiece of tissue 118 as shown in FIG. 17B. For this purpose, the oppositeends 120, 122 of the gel block 110 may be squeezed or compressedtogether to widen the hole 112, with the assistance of the slits 114,116, and the tubular piece of tissue 118 may then be placed in the hole112. Due to the resilience or elasticity of the gel block 110, theopposite sides 124, 126 of the gel block 110 will move back togethertoward the original position and thereby compress or hold the tubulartissue sample 118 in the desired orientation shown in FIG. 17B. The gelblocks illustrated in FIGS. 13-16 , as well as that of FIGS. 17A and17B, are used in the same manner as described above, by sectioning alonga plane defined by the surface of each block that exposes the tissuesample to the microtome blade.

FIGS. 18, 19 and 20 illustrate further examples of gel blocks 130, 140,150 with various types of tissue receiving spaces 132, 142, 152, andcapable of being cut along the dotted lines, for example, such that theuser may customize the tissue sample support structure, i.e., gel blocks130, 140, 150, to his or her needs.

FIGS. 21A, 21B and 21C illustrate another alternative embodiment of agel block 160 that includes elongate recesses 162 along one surface 164for receiving thin threadlike tissue samples 166 as shown in FIG. 21A.As further shown in FIG. 21B, the surface 164, including the tissuesamples 166, may be sprayed with an adhesive material 168 such that acoating 169 is formed over the surface 164 and over the tissue samples166 such that the tissue samples 166 are retained in the recesses orgrooves 162. The adhesive material 168 may be of the type describedabove. The gel blocks 160 with the retained tissue samples 166 are thenused in the above-described manner, for example, with a sectionabletissue cassette 30 (see FIG. 6 ) and with the tissue samples 166 facingthe bottom of the cassette, defining the microtome sectioning plane.Sections are then taken as generally described above in connection withFIG. 6 .

FIG. 22 illustrates another alternative gel block 170 constructed inaccordance with the invention, and including a variety of tissuereceiving spaces 172, 174, 176 for holding tissues of different typesand/or sizes and/or shapes.

FIG. 23 illustrates another embodiment in which two sheet forms 180, 182of the gel compound are used to sandwich tissue samples 184therebetween. The small, thin sheets 180, 182 of the preformed gelcompound may then be embedded in paraffin and sectioned in a microtome.Preferably, at least the sheet 182 that will be sectioned first by themicrotome is as thin as practical so as to minimize the number ofsectioning cuts that are necessary to reach the tissue samples 184. Itwill be appreciated that the surface 182 a carrying the tissue samplesin this example defines the sectioning plane. Another benefit of theinvention is realized when the gel compound structures are used tostraighten or flatten tissue such that a complete and continuous tissuesection is taken by the blade (not shown). For example, if the tissuesamples 184 are wavy and curve out of the plane defined by the surface182 a, then the sandwiching effect of the sheet 180 when placed on topof the tissue samples 184 will straighten or flatten the samples 184into a single plane for effective sectioning.

FIG. 24 illustrates another modification in which the sheet 182 of FIG.23 has been slightly modified into a gel sheet 182′ that includes arecess 190 for containing tissue samples 192, and further includeschannels 194, 196 for expressing or draining fluid, such as formalin, inwhich the tissue samples 192 are stored during transport to a histologylaboratory. In this regard, the histotechnician can pour the vial offluid containing small tissue samples 192 (e.g., shavings, etc.) intothe recess 190 and the fluid can drain through one or more channels 194,196 while leaving the tissue samples 192 in the recess 190. FIG. 25illustrates a use of the gel sheets 180, 182 or 182′ in conjunction witha sectionable cassette 30, as for example, described in FIGS. 6, 12 and21C. As with all figures, like reference numerals are used to referencelike structure and function, and therefore additional description ofsuch common subject matter is not necessary. FIG. 25 provides a clearillustration of another advantage when using a sectionable cassette 30.That is, the bottom sheet 182 prevents the tissue samples 184 fromdirectly contacting the bottom 34 of the cassette 30. If the tissuesamples 184 were to contact the bottom 34, then artifact might beintroduced into the tissue samples 184 due to the discontinuities of thebottom 34 (i.e., its perforated construction). The surface 182 a and thefacing surface 180 a will sandwich the tissue samples 184 and present acontinuous section of the tissue to the microtome blade (not shown)after the bottom 34 and the sheet 182 are sectioned or “faced” off bythe blade during the initial portion of the microtome process.

According to embodiments of the present invention, the gel compounds mayinclude hydrogels, organogels, aerogels, or combinations thereof. Ahydrogel is a network of polymer chains in which water is the dispersedmedium. Exemplary hydrogels include, but are not limited to, siliconhydrogels, proteins based, carbohydrate based or polyol based hydrogels.An organogel is a non-crystalline, non-glassy solid material composed ofa liquid organic phase entrapped in a three-dimensionally cross-linkednetwork. Exemplary organogels include, but are not limited to Lecithinbased and various dendriomer based organogels. An aerogel is a syntheticporous material in which the fluid component of the gel is air or a gas.Exemplary aerogels include, but are not limited to silica based andcarbon based aerogels.

The gel compounds used for constructing the gel blocks disclosed hereinmay be formed in various ways, with two exemplary embodiments beingprovided below. According to embodiments of the invention, the gelcompounds are formed from ingredients, such as macromolecules that arecapable of undergoing cross-linking, cross-linking agents,preservatives, and water or other suitable solvents. Other optionalingredients include dyes, for example.

According to an embodiment of the present invention, the gel compoundincludes hydrogels that include cross-linked macromolecules.Accordingly, the macromolecules are capable of undergoing cross-linking.In one aspect, the macromolecules may contain a plurality of hydroxylgroups, which may react with a suitable cross-linking agent. Exemplarymacromolecules include gelatin, starches such as corn starch, and agars.Other suitable macromolecules include proteins such as serum, albumen,or synthetic polymers like polylysine or polyols. Similarly, manycarbohydrates (e.g., various gums, or cellulose and its derivatives)will also crosslink like corn starch. Characteristics of the gelcompound may differ, particularly in shear strength. Accordingly, theformulations would have to be optimized based on selection of rawmaterials.

Exemplary cross-linking agents such as borax, melamine formaldehyde,sodium aluminate, or potassium tetraborate can be used to produce a gelstructure. According to an embodiment, the cross-linking agent is borax.

Exemplary preservatives include antimicrobial agents, which inhibit moldgrowth. Suitable antimicrobial agents include methyl paraben. Otheranti-microbial agents such as propyl paraben and others can be used.Without antimicrobial agents, the gel compounds may become moldy afterseveral days.

Use of the color is optional. Number of various types of synthetic dyesor other colors can be used. According to an embodiment, a food gradeaqueous color is used during the manufacturing of the gel compound.Various colors can be used as per requirements. One objective of addingcolor to the gel compound is to provide contrast for a technician to beable to easily see cavities made for the tissues in the gel compoundblock allowing for rapid insertion of the tissues into the block therebyimproving the efficiency. The color may wash out during various stagesof tissue processing and staining.

The gel compound further includes water. Deionized or distilled water issuitable, as is tap water. An important factor preparing the gelcompound is temperature of the water. Preferably, the water should becold (e.g., less than about 25° C.), because even lukewarm water willcause a greater degree of clumping. According to an embodiment, thetemperature of the water is between about 5° C. to about 20° C., forexample. Glycol type co-solvents can be used in combination with waterto reduce the water content and shrinkage of the gel due to dryingand/or to modify or incorporate new properties.

Sourcing of the Chemicals

Gelatin comes in different molecular weight ranges (called bloom).Scientific catalogs offer a number of ranges. These have been speciallypurified and sorted, so their cost is high. Grocery store gelatin is abroader range of molecular weights, but at least with name brands, hashigh batch-to-batch uniformity. Gelatin is readily available materialthat makes gel compound with very good flexibility and physicalproperties but gel compound made of gelatin will get stained pink byEosin, which may make it difficult to distinguish the gel from thetissue. Any gelatin can be used, for examples below, Knox brand gelatinwas used. According to one embodiment, the gelatin may be present in thegel compound in an amount ranging from about 2 wt % to about wt %, basedon the total weight of the gel compound.

Starch can be made from a variety of plant sources (wheat, corn, potatoetc.). Corn starch is very inexpensive and readily available inconsistent high quality. Starch from different suppliers or source wouldhave different range of molecular weight, the formula can be optimizebased on raw materials selected. For the examples below, Hulman & Co.'sClabber Girl® household corn starch was used. According to oneembodiment, the starch may be present in the gel compound in an amountranging from about 2 wt % to about wt %, based on the total weight ofthe gel compound.

Agar is a polysaccharide complex (CAS: 9002-18-0) obtained from redalga. Agar is composed of approximately 70% agarose and 30% agaropectin.Agarose is the gel forming part of agar while agaropectin is anon-gelling fraction. Agar was selected over agarose in this applicationdue to cheaper cost of the former. Scientific catalogs offer a number ofranges and modifications of agar mainly for its use as a culture medium.Pure agarose or some of the agar products are specially purified andsorted, so their cost is high. Agar substitutes such as Phytagel™ and/orScleroglucan type materials can be used. Regular average priced agar(Sigma Aldrich Product #A1296) was used for the examples below.According to one embodiment, the agar may be present in the gel compoundin an amount ranging from about 0.1 wt % to about 15 wt %, based on thetotal weight of the gel compound.

Borax is sodium tetraborate. As a mineral it is commonly found as adecahydrate form, but commercially available borax may varysignificantly in its degree of hydration (to improve flow and ease ofsolubility). Brand name (20 Mule Team), household borax was used for theexamples below. The degree of hydration is critical in weighing andshould be considered during formulation. According to one embodiment,the cross-linking agents may be present in the gel compound in an amountranging from about 0.05 wt % to about 5 wt %, based on the total weightof the gel compound.

Methyl paraben is widely available from a number of suppliers. Accordingto one embodiment, the preservatives may be present in the gel compoundin an amount ranging from about 0.05 wt % to about 5 wt %, based on thetotal weight of the gel compound.

As described herein, a sheet of the gel compound is formed in a shallow,nonstick pan. However alternative options such as extrusion may beutilized.

Example 1 Standard Formula (Percentages are w/v Relative to Water)Ingredient w/v Percentage 1× Formula

Ingredient Percentage For 100 sq. in batch size Borax 1.33 1.50 gGelatin 9.33 10.50 g Corn Starch 9.33 10.5 g Methyl Paraben 1.33 0.15 gWater 100 112.50 g

Batch Size

The Standard Formula, or 1× Formula, produces a gel approximately 2 mmthick when poured into a 100 square inch pan. For pans of differentsizes and gels of different thickness, the formula may be scaledproportionally.

Preparation of the Gel Compound of Example 1

Preweighed quantities of corn starch, gelatin and methyl paraben arecombined in a vessel and thoroughly mixed to minimize the formation ofclumps when the water is added. CAUTION: DO NOT ADD BORAX TO THE OTHERSOLID INGREDIENTS. Cold water (e.g., less than 25° C.) is added to themixed dry ingredients without mixing to permit the starch to hydrate forabout a minute or longer. Thoroughly blend the aqueous mixture to ensurethat substantially all of the corn starch, gelatin, and methyl parabenare evenly dispersed therein. Heat the aqueous mixture until it startsto boil, stirring or otherwise mixing periodically throughout theprocess. After which, the heating of the aqueous mixture is discontinuedand the preweighed quantity of borax is added while mechanically mixingfor a few seconds until the borax is completely dispersed.

Working as fast as possible, the resultant material is poured into ashallow, non-stick pan. The pan is tilted in all directions to get thematerial to flow into all corners and edges, then the pan is set downlevel and gravity is allowed to bring the material to uniform thickness.The pan is covered with plastic wrap and the material is allowed to coolto room temperature without moving to form a sheet of gel compound.After 2 or more hours, the sheet of gel compound is removed from the panin one piece. For example, the sheet may be removed from the pan byprying it up along one edge with a spatula or similar device, thenpulling it up and out. The sheet of gel compound is placed on a smoothpiece of plastic wrap and cut. The gel is cut into 12×18 mm blocks (orin required size). Slits or holes desired for supporting specific tissueshapes are then created in the blocks. Required shape and cavities forsupporting tissues can be achieved using molds during processing or bydie cutting post manufacturing.

Chemical Explanation:

Gelatin produces a strong gel when processed properly. In hydrated format room temperature, its molecules are tightly wrapped balls that do notinteract with each other (or with other ingredients), minimizingclumping. The dispersion is somewhat viscous but will not gel as is. Astemperature is raised to the boiling point, the molecules unwrap andbecome long, tangled strings. Upon cooling, they retain the tangledconformation and become a sponge-like mass entrapping water. A puregelatin dispersion is reversibly gel-like or liquid, depending upon thetemperature.

Corn starch is also a macromolecule with similarities to gelatin. Ittends to clump badly when put into cold water because of stronginteractions between adjacent molecules. When dry, starch and gelatinparticles stick to one another and minimize starch-starch interactionsduring hydration, hence the need to mix dry ingredients together. Likegelatin, starch molecules unwind at higher temperature and form a softgel upon cooling. Starch is used as a thickener, but at highconcentrations, it forms a gel with little shear strength. The functionof starch in the gel is to provide reactive hydroxyl groups forcrosslinking.

Borax is a crosslinking agent that reacts with the hydroxyl groups foundin carbohydrates like starch. Crosslinking makes the gellingirreversible. The reaction is rapid initially, so the gel must be pouredimmediately after incorporating borax into the mixture.

Borax and starch alone will produce a permanent gel, but a combinationof the two was used to obtain physical properties such as shear strengthrequired in the purposes in this application.

Example 2

Standard Formula (Percentages are w/w)

Ingredient w/w Percentage 1× Formula

Material Percentage For 100 sq. in batch size Agar or agarose 2.00 2.70g Borax 0.50 0.68 g Corn Starch 3.00 4.05 g D.I. Water 94.30 127.30 gFood grade dye 0.05 0.07 g Methyl Paraben 0.15 0.20 g

Batch Size

The standard batch size produces a gel approximately 2 mm thick whenpoured into a 100 square inch pan. For using pans of different sizes orobtaining gels of different thickness, the formula should be scaled upor down proportionally.

Preparation of the Gel Compound of Example 2:

Weight of empty container to be used for making the gel is measured andrecorded. Preweighed quantities of corn starch, agar and methyl parabenare combined in the container and thoroughly mixed to minimize theformation of clumps when the water is added. CAUTION: BORAX MUST NOT BEADDED TO THE OTHER DRY INGREDIENTS. Cold water (e.g., less than 25° C.)is added. The aqueous mixture is thoroughly blended to ensure thatsubstantially all of the corn starch, gelatin, and methyl paraben areevenly dispersed therein. Food grade dye of the desired color is addedto the aqueous mixture and mixed well to disperse dye and to provide ahomogeneous color to the mixture. The aqueous mixture is heated until itstarts to boil, stirring or otherwise mixing periodically throughout theprocess. After which, the heating of the aqueous mixture isdiscontinued. The container and its contents are weighed. If necessary,water is added and mixed to replenish and compensate for water loss informulation due to evaporation. The preweighed quantity of boraxis isadded and the aqueous mixture is stirred for a few seconds until theborax is completely dispersed/dissolved.

Working as fast as possible, the resultant material is poured into ashallow, non-stick pan. The pan is tilted in all directions to get thematerial to flow into all corners and edges, then the pan is set downlevel and gravity is allowed to bring the material to uniform thickness.The pan is covered with plastic wrap and the material is allowed to coolto room temperature without moving to form a sheet of gel compound.After providing adequate gelling time, the sheet of gel compound isremoved from the pan in one piece. For example, the sheet may be removedfrom the pan by prying it up along one edge with a spatula or similardevice, then pulling it up and out. The sheet of gel compound is placedon a smooth piece of plastic wrap and cut. The gel is cut into 12×18 mmblocks (or in required size). Slits or holes desired for supportingspecific tissue shapes are carefully created in the gel block. Requiredshape and cavities for supporting tissues can be achieved using moldsduring processing or by die cutting post manufacturing.

Chemical Explanation

Agar (or agarose) produces a strong gel when processed properly. In adry powder form, its molecules are tightly wrapped particles that do notinteract with each other (or with other ingredients), minimizingclumping. Its water dispersion is somewhat viscous but not a gel. Astemperature is raised to the boiling point, the molecules unwrap andbecome long, tangled strings. Upon cooling, they retain the tangledconformation and become a sponge-like mass entrapping water. A pure agar(or agarose) dispersion makes a reversible gel with physical propertiesand stability depending upon the temperature.

Corn starch is also a macromolecule with similarities to agar. However,it tends to clump badly when put into water because of stronginteractions between adjacent molecules. When dry, starch and agarparticles stick to one another and minimize starch-starch interactionsduring hydration, hence the need for mixing the dry ingredients togetherprior to hydration. Like agar, starch molecules unwind at highertemperature and form a soft gel upon cooling. Starch is used as athickener, but at high concentrations, it forms a gel with little shearstrength. Its function in the gel compound described herein is toprovide reactive hydroxyl groups for crosslinking and optimize physicalproperties of the gel.

Borax is the crosslinking agent that reacts with the hydroxyl groupsfound in carbohydrates like starch or agar. Crosslinking permanentlysolidifies the gel. The reaction is rapid initially, so the gel must bepoured rapidly after incorporating borax into the mixture.

Borax and either agar or starch alone will produce a permanent gel, butthese binary compositions (borax/agar or borax/starch) are used toobtain physical properties such as shear strength required for thepurposes of this application.

Agar is an important component in the embodiments described herein. Oneof the preferred reasons for using of agar over other gel formingmaterials such as gelatin is that agar does not get stained by Eosin.Hence during microscopic evaluation the tissues can be very easilydistinguished from surrounding gel.

Methyl paraben is an antimicrobial agent. Without it, gels become moldyafter several days.

Water is the largest component of this formulation. Deionized ordistilled is suitable, as is tap water. An important factor in preparingthe gel compound is temperature of the water. Preferably, the watershould be cold (e.g., less than about 25° C.), because even lukewarmwater will cause a greater degree of clumping. The water should be coldwhen first added into starch-agar mixture, because warm water will causea greater degree of clumping of the corn starch, which makes the initialmixing difficult.

Variations in Manufacturing Process

Order of addition of initial dry materials (e.g., agar, corn starch andmethyl paraben), as well as water, can be changed as per comfort ofmanufacturing process.

Materials can be heated in a conventional manner or using microwaves. Ithas been found that microwave heating is especially suitable for heatingsmall batches. More specifically, it has been observed that the time forheating reduces significantly and more homogeneous heating is obtainedwith without material settling and sticking to the bottom of thecontainer.

Variation in proportions of raw materials like agar, corn starch, borax,water, etc. may affect the physical properties of the gel to differingdegrees. However, a gel with workable physical properties can beobtained over a considerable range of reactants. The formulationspresented in Examples 1 and 2 have been found to provide improved batchto batch consistency under the described manufacturing processes.

While the present invention has been illustrated by the description ofone or more embodiments thereof, and while the embodiments have beendescribed in considerable detail, they are not intended to restrict orin any way limit the scope of the appended claims to such detail. Thevarious features as described herein may be used in the describedcombinations or in any combination according to the needs of the user.Additional advantages and modifications will readily appear to thoseskilled in the art. The invention in its broader aspects is thereforenot limited to the specific details, representative apparatus and methodand illustrative examples shown and described. Accordingly, departuresmay be from such details without departing from the scope or spirit ofthe general inventive concept.

What is claimed is:
 1. Apparatus comprising: a three dimensional,self-supporting and preformed gel block having a predetermined geometricshape defined by an outer boundary for retention and orientation of atleast one tissue sample during a histopathology process includingprocessing, embedding and microtome slicing of the tissue sample, thegel block being sectionable by a microtome to form ribbon-like sectionsof the gel block and tissue sample; preformed tissue retaining structurecomprising a tissue sample receiving space formed in a portion of thethree dimensional, self-supporting and preformed gel block, thepreformed tissue sample receiving space formed by the gel block andconfigured to retain and orient the tissue sample, and a packageenclosing the gel block under clean, moisture-proof conditions.
 2. Theapparatus of claim 1, wherein the gel block is resilient such that afterdeformation from an original shape, the gel compound reverts back to theoriginal shape.
 3. The apparatus of claim 1, wherein the tissuereceiving space comprises at least one of: a preformed slit in the gelblock, a preformed hole formed in the gel block, a preformed recessformed in the gel block, or combinations thereof.
 4. The apparatus ofclaim 1, wherein the tissue retaining structure further comprises atleast one deformable portion of the gel block configured to apply aforce to the tissue sample and thereby retain the tissue sample in adesired orientation.
 5. The apparatus of claim 4, wherein the deformableportion further comprises a hinged jaw element configured to movebetween open and closed positions and apply a clamping force to thetissue sample in the closed position to thereby maintain the tissuesample in a desired orientation.
 6. The apparatus of claim 1, whereinthe gel block is permeable to fluids and reagents used in processing thetissue sample to thereby provide full cross-sectional preservation ofthe tissue sample.
 7. A method of orienting, processing, embedding andmicrotome slicing a tissue sample using a gel compound preformed into athree dimensional, self-supporting and preformed gel block having apredetermined geometric shape defined by an outer boundary for retentionand orientation of at least one tissue sample during a histopathologyprocess including processing, embedding and microtome slicing of thetissue sample, the gel block being sectionable by a microtome to formsections of the gel block and tissue sample, the gel block including atissue sample receiving space formed in a portion of the threedimensional, self-supporting and preformed gel block, the methodcomprising: removing the preformed gel block from a package enclosingthe gel block under clean, moisture-proof conditions; retaining thetissue sample in a desired orientation in the tissue sample receivingspace; processing the tissue sample while in the desired orientation bysubjecting the tissue sample and the preformed gel block to processingfluids and reagents; embedding the preformed gel block and the tissuesample while in the desired orientation in an embedding media to form amicrotome sectionable block of the embedding media, the tissue sampleand the preformed gel block; and microtome sectioning the microtomesectionable block to obtain the sections of the tissue sample fordiagnosis.
 8. The method of claim 7, wherein retaining the tissue samplefurther comprises retaining the tissue sample between resilientlydeformable portions of the preformed gel block.
 9. The method of claim7, wherein retaining the tissue sample further comprises retaining thetissue sample in a through hole formed in the preformed gel block andapplying force to the tissue sample with the gel block.
 10. The methodof claim 7, further comprising: securing the preformed gel block and theretained tissue sample on a microtome sectionable tissue supportstructure at least prior to the embedding and microtome sectioningsteps; and microtome sectioning the tissue support structure before thestep of microtome sectioning the microtome sectionable block.
 11. Themethod of claim 10, wherein the microtome sectionable tissue supportstructure further comprises a cassette including first and secondportions, and the method further comprises: securing the gel block andthe retained tissue sample between the first and second portions of thecassette; and microtome sectioning through at least one of the first orsecond portions.
 12. The method of claim 7, wherein retaining the tissuefurther comprises using an adhesive to secure the tissue sample to thegel block.